Experiments in this R21 application are designed to analyze protein expression in cardiac samples collected from failing human hearts using a powerful multi-dimensional mass spectrometry (MS) approach. The overall aim of this study is to identify proteins with large scale changes in expression, and then focus on protein expression of classical protein kinase C (PKC) isoforms and proteins associated with these isoforms in failing human hearts. An existing set of samples will be analyzed to compare protein expression in failing versus non-failing hearts. Protein expression in response to mechanical unloading by ventricular assist device therapy and alterations in regional expression of proteins also will be evaluated in failing human hearts. A quantitative proteomic strategy will be used to identify proteins with large-scale changes in expression using liquid phase chromatofocusing, reverse phase HPLC and electrospray time-of-flight tandem mass spectrometry. Protein expression of classical PKC isoforms and associated proteins will be analyzed by protein microarray analysis of these fractions. Results from these studies are expected to provide insight into the changes in global and PKC protein expression during human heart failure, and identify key proteins and future therapeutic targets to explore in animal models of heart failure. PUBLIC HEALTH RELEVANCE Analysis of the large scale changes in protein expression and in the expression of protein within the PKC signaling cascade are expected to provide new insights into some critical changes in protein expression that occur with human heart failure. These experiments will provide fundamental information needed to test new hypotheses in animal models of heart failure, and design future therapeutic targets for treating heart failure.